The goal of this program is to continue developing our novel antimicrobial, Novo29, into a therapeutic for treating a wide range of infections caused by Gram-positive pathogens including Staphylococcus aureus, Streptococcus pneumoniae, and Bacillus anthracis. Using our iChip culturing technology, NovoBiotic has been exploiting previously uncultivable bacteria that make up 99% of all environmental microorganisms for production of new antibiotics. In 2015, NovoBiotic reported the discovery of teixobactin, the first member of a novel class of peptidoglycan synthesis inhibitors. We have since discovered Novo29, yet another promising and potent inhibitor of Gram-positive pathogens. Like teixobactin, Novo29 rapidly kills bacteria by inhibiting bacterial cell wall synthesis. However, Novo29 is significantly smaller than teixobactin, shows different target binding characteristics, and does not have serum gelation issues that require special formulations. Novo29 showed excellent efficacy against MRSA in two mouse models of infection (septicemia and thigh infection). The compound showed promising pharmacokinetic properties in mice, and is metabolically stable when incubated with liver microsomes. No spontaneous resistance (<10-10) occurred against S. aureus, and serial passaging in sublethal concentrations of drug failed to generate resistance. All these results demonstrate promising potential for Novo29 to treat drug-resistant infections. The goal of this project is to continue advancing Novo29 through preclinical research in preparation for IND-enabling studies. This work will include: (Aim 1) produce enough compound through fermentation to conduct all proposed studies in this grant, and for IND-enabling studies; (Aim 2) establish expanded MIC90s for pathogens isolated from recent clinical infections. Determine MBCs, time-kill profiles and post antibiotic effect (PAE) against relevant pathogens; (Aim 3) continue evaluating toxicity and pharmacokinetic (PK) profiles in animal models. Identify and characterize any major microsomal metabolites. Evaluate drug-drug interactions, genotoxicity, and cardiac safety in in vitro assays. Determine which PK/PD parameter(s) best correlate with efficacy; (Aim 4) examine efficacy of Novo29 in animal models of pulmonary infection with MRSA and anthrax; (Aim 5) explore the structure activity relationship (SAR) of Novo29 through medicinal chemistry. Design and test back-up analogs that may have increased potency and oral bioavailability. At the conclusion of this grant, Novo29 will be prepared to enter formal IND-enabling studies and advance to the clinic.